Work package 3: Chemical analysis, morphology, and concentration of MV

The aim of this work package is to develop methods to distinguish MV from other biomaterials and to identify the type of MV by analysing the chemical composition and morphology of MV. In addition, methodologies to determine the concentration of MV will be developed.

Human plasma does not only contain MV, but also similar sized contaminants, such as lipoproteins and small platelets. Since contaminants are likely to differ in chemical composition from MV, contaminants can be potentially distinguished from MV by chemical analysis. In addition, since MV originate from different cell types, MV contain elements which are characteristic of their parental cell and cell state. The presence of elements like selenium, phosphor, iron, potassium, sodium, and calcium, specific enzymes or proteins, and parts of DNA or RNA inside MV may be characteristic for one specific cell type. In other words, by means of chemical analysis, the cellular origin of MV can be revealed. Thus far, very little is known about the elemental composition of MV.

Supplementary to the chemical analysis is the morphology of MV. With morphology we mean the shape and ultrastructure. Different morphologies can be distinguished when MV are imaged with techniques having sub-nanometer resolution, A validated classification of the different type of morphologies has however never been made, and it is unknown whether the morphology of an MV correlates with its cellular origin, which would be very relevant from clinical point of view, but also for monitoring purification protocols.

Since for many diseases the concentration of MV in plasma from patients is increased, the concentration of MV is of clinical relevance. In addition, a reliable determination of the concentration of MV would improve isolation protocols. However, methods that are able to obtain the concentration of MV directly in suspension are insufficient, since their sensitivity is limited, such that the concentration of relatively small MV is unknown. On the other hand, techniques that are capable of imaging even the smallest MV only provide the minimum concentration, since the binding efficiency of MV to the surface is unknown.

In WP3, the elemental composition of an ensemble of MV will be determined by chemical analysis using Anomalous Small Angle X-ray Scattering (ASAXS) and X-ray fluorescence (XRF). By way of functionalized tips with Atomic Force Microscopy (AFM), specific enzymes or proteins on the outside of MV could possibly be detected. The application of functionalized probe and appropriate antibodies to coat the probe will be investigated. Furthermore, the morphology of MV will be imaged by Transmission Electron Microscopy (TEM) and AFM with sub-nm resolution and possible links between observed characteristics and origin of the MV will be investigated. In addition, the determination of the concentration of MV is carried out.